Many therapeutic agents in use today are small molecule natural products produced by bacteria. While their use in clinical settings has been a boon for medicine, the functions that these molecules serve for the producing organisms are not well understood. This gap in knowledge may hinder efforts at discovering new compounds that may be developed into effective therapeutics in the future. Initially thought to serve primarily as agents of mutual destruction, we now recognize that many microbial products serve as signals that microbes sense in order to adapt to their environment. This ecological perspective of the role of natural products led to the development of our central hypothesis: Bacteria synthesize and secrete a large number of small signaling molecules that affect the physiology of other microbes that occupy the same habitat. This general hypothesis drives our proposed research. But the lines of experimentation we follow are also discovery-driven; over the past few years we have discovered new small molecules that mediate diverse interspecies interactions in the microbial world. In the initial phase of this project, we validated the main concept that studying the chemical biology of interspecies interactions can lead to the discovery of new small molecule natural products. As before, we will continue to follow approaches that meld the disciplines of microbial ecology, physiology, and genetics, with enzymology, bioinformatics, and small molecule chemistry. We now aim to take this multidisciplinary approach to the next level by raising the scale of our screens and increasing the throughput of our compound characterization. Specifically, we will pursue research along three directions aims: (i) We will carry out high-throughput, broad spectrum screens to discover molecules mediating interspecies interactions. (ii) We will investigate specific ecological microbial interactions to discover molecules that mediate them. (iii) We will carry out pair-wise co-cultures of actinomycete strains whose genomes have been sequenced to carry out genome assisted compound discovery. Throughout our analyses, we will use diverse methodologies ranging from Nanostring assays of transcriptional effects to high-throughput liquid chromatography/mass spectrometry and imaging mass spectrometry in order to characterize the nature of the interactions and to define the molecules involved. PUBLIC HEALTH RELEVANCE: The development of antibiotics as therapeutics is one of the key accomplishments of modern medicine. Penicillin (the first antibiotic to be intensively used) and many other antibiotics, many anti-cancer agents, and many immunosuppressants, are molecules produced by microorganisms, they are thus called small molecule natural products. Unfortunately, the appearance of antibiotic resistant bacteria has diminished the effectiveness of existing antibiotics and there is always a great need to discover more of these molecules; the proposed research aims at discovering new such molecules by studying the effects they have in mediating interactions among microbes.